Alopecia areata (AA) has been suspected to be an autoimmune disease, although there is no distinct evidence, we investigated the relationship between AA and autoantibodies against dense fine speckles 70 kDa (DFS70) in 111 patients with alopecia and 105 healthy controls. The sera from 59 out of 111 (53%) Japanese alopecia patients were positive for anti-nuclear antibody (ANA), as compared to the sera of 16 out of 105 (15%) healthy controls (p < 0.001). Twenty percent (22/111) of the alopecia patients were shown to be positive for the prevalence of anti-DFS70 antibodies, as compared to 8% (8/105) of the healthy controls (p < 0.01). IgG subclass analysis by ELISA showed that IgG1 and IgG2-anti-DFS70 antibodies were dominant in alopecia patients. The DFS70 gene expression in the hair structures was clearly detected in both those with and those without the anti-DFS70 antibody by RT-PCR. Immunohistochemical techniques showed that the DFS70 was localized predominantly in the outer root sheath (ORS) cells. The elevated anti-DFS70 antibodies in alopecia patients and the localization of the DFS70 in the ORS suggest that autoantibodies against the DFS70 are related to the etiology in a certain population of AA.